植物激素对石刁柏花药培养中染色体倍性变异的影响

在石刁柏花药培养过程中,2,4-D在引起愈伤组织细胞染色体倍性变异方面起主导作用,高浓度的2,4-D虽然能提高愈伤组织中单倍体细胞的频率,但同时又加剧其多倍化。不同激素配比的分化培养基在诱使再生植株染色体倍性变异方面存在着明显差异。在附加1.0ppmBA和0.2ppmIBA的培养基上,再生植株茎尖单倍体细胞频率最高,达31.17％。比较愈伤组织和再生植株的染色体倍性变异幅度,发现后者明显地大于前者。     

燕麦不同品种染色体倍性的检测

研究了6个不同品种燕麦的染色体倍性差异,通过根尖染色体计数法和气孔保卫细胞横纵径及保卫细胞叶绿体计数法等方法对燕麦染色体倍性进行了观察鉴定.结果表明,6个不同品种燕麦中定燕1号、晋燕8号、晋燕17号、宁莜1号为六倍体(2 n=6 x=42),白燕6号、白燕7号为四倍体(2 n=4 x=28),同倍性不同种之间气孔保卫细胞叶绿体数目相近,不同倍性间气孔保卫细胞的大小差异较明显.经X2适合性检测,六倍体和四倍体植株气孔保卫细胞横径均值之比和保卫细胞叶绿体数的平均值之比符合其染色体数目之比3∶2,而纵径均值比值不符合.     

石刁柏花药离体培养及单倍体植株再生的研究

1988至1990年进行了石刁柏花药离体培养,将采集不同时期花蕾接种,低温处理,激素配比,花药培养过程中形态学及花粉粒发育途径,愈伤组织诱导及其类型,花药愈伤组织再分化和染色体倍性,单倍体植株的再生及染色体加倍后入土移栽等方面,均做了较深入的研究,并获得了超雄株即纯合二倍体。     

水稻多倍性育种中的倍性鉴定方法研究

从水稻花粉粒、植株形态、种子形态和染色体计数等方面分别对不同倍性水稻植株的染色体倍性进行了鉴定.鉴定结果表明,随着细胞染色体倍性的增加,植物细胞和器官体积一般趋于增大,水稻花粉粒大小与染色体数目和倍性存在正相关性,可作为鉴定水稻倍性的参考指标;水稻植株形态和籽粒大小、芒的有无、着粒密度和结实率高低等性状与染色体倍性同样具有相关性,亦可作为鉴定水稻倍性的参考指标.     

不同倍性苎麻的细胞学观察

化学药剂诱导的苎麻孤雌生殖后代植株的染色体数目变化很大, 有单倍体、 混倍体和非整倍体细胞。 在28株孤雌生殖后代植株中, 有6株单倍体细胞超过50%。 秋水仙素诱变株及多倍体杂交后代的染色体数目也呈现多样性, 不同植株甚至同一植株的染色体数目均不一致, 变化在12～84之间。 对不同倍性苎麻植株分生组织的有丝分裂行     

萝卜花药培养技术研究

通过单倍体育种途径可快速获得纯合材料,增加有益性状的选择几率,加快育种进程。花药培养是获得单倍体的有效途径之一,已在十字花科芸薹属作物上取得巨大成功。但关于萝卜花药培养的报道极少。以15个不同类型的萝卜品种为试材,研究了花药离体培养中胚状体的诱导及再生。结果表明:基因型是限制花药培养成胚的关键因素,15个品种中,有4个品种获得了胚状体及再生苗。同时植株生长条件也对胚状体的形成产生影响。采用流式细胞仪(FCM)鉴定了再生株的倍性,再生株群体的倍性组成比较复杂,以二倍体为主。     

滇丁香化学诱变的多倍体及其鉴定研究

为获得滇丁香新种质、选育抗性新品种,用不同浓度的氨磺灵溶液浸泡滇丁香种子,以种子下胚轴膨大视为变异株,统计变异率和发芽率,通过形态学观察、气孔观察、染色体计数及流式细胞仪检测确定其倍性。结果表明：15μmol/L氨磺灵浸泡种子24 h为滇丁香多倍体诱导最佳方法,变异率达60.1%,发芽率为66.7%,获得2株四倍体植株;25μmol/L氨磺灵诱导24 h,变异率为50%,获得1株八倍体植株。滇丁香变异植株在株高、叶长、叶厚方面均与对照植株呈显著差异,多倍体滇丁香植株株型矮小、叶片畸形较圆润且变厚、叶色较深、叶毛茎毛粗长。多倍体植株气孔的长和宽及保卫细胞的长和宽均显著大于二倍体植株,且气孔密度显著小于二倍体植株。经流式细胞仪检测,不同倍性植株DNA含量近似倍数关系。经染色体计数,滇丁香二倍体植株染色体数目为2n=44,四倍体植株4n=88。四倍体滇丁香丙二醛含量极显著低于二倍体,而脯氨酸含量极显著高于二倍体。     

基因型对陆地棉花药离体培养反应的影响

对２７种棉花基因型的花药在离体培养中的反应进行了比较,结果表明：所有的基因型在合适的培养条件下均能高频率地形成愈伤组织,但不同基因型间愈伤组织诱导率和诱导量不同;再生植株的花药比原始亲本的花药易诱导出愈伤组织;品种间杂种表现出部分优势,但不明显。本试验中,仅鲁棉６号和Ｓｉｏｋｒａ１－３两个品种诱导获得了胚性愈伤组织、胚状体和再生植株,且二者之间也存在着差别。从基因型在棉花花药培养中的效应、外界调控对棉花不同基因型花药培养效果的影响、棉花花药培养与体细胞培养基因型差异的比较等三个方面进行了讨论,提出了棉花花药培养愈伤组织诱导和植株再生是受两套遗传基因控制的遗传性状,并提出了棉花组织培养植株再生的阈值问题     

甘蓝未受精子房离体培养及再生植株倍性的鉴定

甘蓝通过未受精子房离体培养诱导获得的再生植株,对再生植株的倍性进行有效的鉴定是将其进一步应用于优良品种选育的基础。本研究利用3种基因型的甘蓝材料(PMQM、QMF、RMQM)培育再生植株,优化甘蓝未受精子房离体培养体系,并通过形态学鉴定法、根尖染色体计数法、流式细胞仪鉴定法对组培植株进行倍性鉴定。结果表明:在0.4 mg/L ZT的分化培养基中,3种基因型材料的愈伤组织分化率明显高于1.0 mg/L 6-BA培养基中的组培苗,其中基因型RMQM的分化效果最好;最终确定诱导愈伤组织分化不定芽的最适培养基配方为MS+0.4 mg/L ZT+2.0 mg/L 2,4-D+0.1 mg/L NAA,且通过3种鉴定方法,得出再生植株倍性:单倍体3.4%,双倍体49.8%,四倍体15.9%,嵌合体35.3%。     

顶坛花椒组织培养再生体系的建立

为建立顶坛花椒组培再生体系,以贵州地方品种顶坛花椒的未成熟果实为外植体,研究不同浓度细胞分裂素(6-BA)和生长素(2,4-D)组合对珠心组织诱导的影响,并对其再生培养过程进行了研究。结果表明,MS+6-BA 2.0 mg/L+2,4-D 0.4 mg/L为珠心组织初期培养和胚状体诱导的最佳培养基,WPM+6-BA 0.5 mg/L+IBA 0.1 mg/L为最佳分化培养基,1/2WPM+IBA 0.2 mg/L为生根培养基。以流式细胞仪对再生植株进行染色体倍性分析,结果表明珠心组织再生植株与母株染色体倍性一致,均为二倍体。     

The Use of Stomatal Chloroplast Number for Rapid Determination of Ploidy Level in Maize

The usefulness of using the chloroplast number in epidermal guard cells as an indirect ploidy indicator was evaluated on seed-grown and tissue culture-derived maize plants. For seed-grown plants, two maize genotypes (B89 and R75) which had both diploid and tetraploid seeds available were used as experimental materials. The ploidy levels of seed-grown plants were confirmed by flow cytometric analysis of nuclear suspensions from these plants. For regenerated plants, haploid and diploid levels were examined and the ploidy levels of these plants were determined by chromosome counts of cells from root tips. A positive relationship between the chloroplast number and ploidy level was observed for both seed-grown and regenerated plants. The stomatal guard cells of tetraploid plants had nearly double the number of chloroplasts as the diploid plants. Similar results were found from the regenerated plants. The differences in the mean chloroplast number between diploid and tetraploid seed-grown plants and between haploid and diploid regenerated plants were highly significant. The results of this study demonstrate that counting chloroplasts in guard cells can be an efficient means of screening a large number of plants for ploidy levels. In addition, this study suggests the possibilities of using this method for detecting contaminated seed lots by different ploidy seed and for distinguishing plants of different genotypes.     

Analyses of phenotype and ARGOS and ASY1 expression in a ploidy Chinese cabbage series derived from one haploid

The aim of this research was to improve our understanding of how ploidy level influences phenotype and gene expression in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Haploid plants (2n = 10) was induced by 0.2% colchicine to produce diploid (2n = 20) and tetraploid plants (2n = 40). The aneuploid (2n = 24) was also obtained by hybridization between diploid plants as the female and tetraploid plants. The ploidy levels of all plants were identified through chromosome counts and flow cytometry. Leaves and petals became larger as the ploidy level increased from haploid to diploid, and from aneuploid to tetraploid. Similarly, expression of ARGOS was regulated by genome size, increasing in parallel with the level of ploidy. Among the four ploidy types, expression was stronger in the floral buds than in the leaves. Expression by ASY1 also differed according to ploidy level, being highest in diploid plants, followed in order by tetraploids. Expression was similar between haploids and aneuploids at two stages—prior to and after meiosis—but was higher in the haploids during meiosis. When buds were compared within the same ploidy type at different stages, ASY1 expression was obviously higher during meiosis than either before or after. Our study demonstrated the generation and phenotype of a ploidy Chinese cabbage series derived from one haploid. Expression of genes ARGOS and ASY1 were modulated by genome size in this ploidy series, and the regulated patterns of the two genes was different.     

Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)

We present an improved protocol for highly efficient production of doubled haploid loose-curd cauliflower plants (Brassica oleracea var. botrytis) via microspore culture. Our experiment explored factors such as donor plant treatment, flower bud pretreatment, embryo germination medium, and ploidy characterization of regenerated plants. Our technique efficiently produced embryos from both tight- and loose-curd donor plants, although the embryo yields were genotype dependent. We achieved a germination rate of around 30 % by employing a hormone combination of zeatin, indole-3-acetic acid, and 6-benzylaminopurine pretreatment culture. We also used 1–4 days of cold pretreatment of the flower buds, which were submerged into NLN-13 medium, to induce microspore embryogenesis. Analysis using an FCM Ploidy Analyzer showed that more than 50 % of regenerated plants were spontaneously doubled haploids, more than 25 % were tetraploids, and fewer than 7 % were haploid. Visual examination of plants in the field revealed that they had distinct phenotypic characteristics relating to their ploidy level. The efficient production of double haploids using our improved microspore culture technique is a promising approach that can be applied in loose-curd cauliflower breeding programmes and genetic research.     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant.     

中国水仙花药培养及植株再生体系建立

本研究以中国水仙花药为外植体,通过器官发生途径建立其植株再生体系,并通过染色体计数鉴定筛选变异个体。结果显示:在4℃下预处理3d有利于花药愈伤组织的形成;愈伤组织诱导培养基最适配比为:MS+2,4-D1.0mg/L+BA0.5mg/L+CH500mg/L+AC500mg/L;愈伤组织分化小鳞茎的最适培养基为:MS+BA0.5mg/L+NAA0.1mg/L+CH500mg/L+AC1000mg/L。通过染色体计数对38个再生植株进行倍性鉴定,结果显示其中30个为三倍体(2n=30),8个为非整倍体(2n=10,11,12,14,17,26)。以这些再生苗为外植体,经器官发生途径,建立了不同倍性的再生体系。     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

Comparison of Anther Culture and the Hordeum bulbosum Method for the Production of Doubled Haploids in Winter Barley

The comparison, between the efficiency of anther culture and the Hordeum bulbosum method in barley was extended to the chromosome number distributions of all planes derived from the two techniques and the proportions of fertile doubled haploid plants which survived until maturity. The frequencies of haploid and spontaneously doubled haploid plants which were useful for practical breeding purposes were found to be around 90 % for both techniques. The remainder consisted of polyploid, mixoploid and aneuploid variants in the case of microspore-derived plants and diploid interspecific hybrids in the progeny of the H. bulbosum method. The ploidy level distributions of the microspore- and H. bulbosum-derived plants appeared to be independent of the genotype of the donor. There were no significant differences between techniques regarding the proportions, of plants which survived a severe winter and the production of fertile doubled haploid plants. Both techniques can therefore complement each other in a breeding programme and their relative merits are discussed. Possible ways of improving doubled haploid production in barley are suggested for increasing its use in breeding schemes.     

组织培养中大葱染色体倍性变异的研究

采用大葱茎尖分生组织直接成苗及分化丛生苗、茎盘诱导愈伤组织培养再生植株,通过染色体压片,对大葱愈伤组织及幼苗染色体数目变化进行了研究。结果表明,茎尖分生组织培养的幼苗及丛生苗遗传稳定,其染色体未发生倍性变异,均为2n=16;愈伤组织及其再生苗遗传稳定性较差,愈伤组织染色体数变异率为43.4%,其中单倍体占6.7%、三倍体占2.5%、四倍体占10%、五倍体占4.2%、六倍体占3.3%、七倍体占4.2%、八倍体占3.3%、非整倍体占9.2%;愈伤组织分化苗染色体变异率为11.7%,其中单倍体占6.7%,三倍体占1.7%,四倍体占3.3%。     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.